In recent years, highly pure protein reagents or protein drugs are highly demanded for researches in the biotechnology-related field or in the pharmaceutical industry. In a method of producing a highly pure protein, purification processes based on various principles such as hydrophobic chromatography, gel filtration chromatography, and ion exchange chromatography may be employed. Affinity chromatography is a particularly excellent method because of being capable of separating a target protein from foreign substances and of concentrating the protein at remarkably high efficiency. In the affinity chromatography, the target protein is separated from foreign substances by adsorbing the protein on an insoluble carrier including, as a ligand, a molecule having specific affinity for the protein, and the protein is used as the ligand molecule in some cases.
Protein A is a protein derived from Staphylococcus aureus and has a repeated structure including five immunoglobulin-binding domains having homology to each other, called E-domain, D-domain, A-domain, B-domain, and C-domain. An immunoglobulin-binding domain is known to bind singly to an immunoglobulin (Non-Patent Document 1), and a recombinant protein including only immunoglobulin-binding domains having a partially-modified amino acid sequence is widely used as the affinity ligand for the affinity chromatography, along with a natural protein A. The Staphylococcus is known to express a protein capable of binding to an immunoglobulin other than the protein A (Patent Document 1), and a partial structure of the protein other than the protein A includes a region in an amino acid sequence having about 45% homology to an immunoglobulin-binding domain of the protein A. However, the availability of this immunoglobulin-binding domain as the affinity ligand, such as chemical stability under acidic and alkaline pH conditions, is unknown.
As for the protein A, in order to provide an adsorptive medium having chemical stability, which is at least equal to or higher than that of another adsorbent based on bonding specificity to IgG, a bonding amount, and the natural protein A, there has been suggested a technique for using, as the ligand, the protein A (rProtein A cys) where one cysteine residue has been introduced and immobilizing the protein A via a thioester bond on an affinity carrier at one point. Moreover, in order to solve problems of the chemical stability on an immobilization site, which is an issue of an affinity carrier for antibody purification such as affinity chromatography carrier where the Protein A cys has been immobilized and problems of sterilization/washing steps due to the chemical stability, and in order to adsorb antibody molecules in larger amounts, there has been suggested a technique for immobilizing the molecules in a C-terminal carboxyl group selective manner via an amide bond on a carrier where a polymer compound having a primary amino group has been introduced (Patent Document 3).
Patent Document 4 further suggests: an immunoglobulin-binding protein that can, for example, be derived from a protein capable of binding to other regions of the immunoglobulin molecule other than the complementary determining regions (CDR), such as B-domain of Staphylococcal protein A, in which at least one asparagine residue has been mutated to an amino acid other than a glutamine or aspartic acid, which mutation confers an increased chemical stability at pH-values of up to about 13 to 14 compared to the parental molecule; and a matrix for affinity separation, which includes the protein as a ligand coupled to a solid carrier. The best embodiment for imparting alkali resistance shown in this document is a combination of substitution of a threonine for the aspartic acid at position 23 and substitution of a glutamic acid for the asparagine at position 43.    Patent Document 1: U.S. Pat. No. 6,548,639 B1    Patent Document 2: U.S. Pat. No. 6,399,750 B1 (JP 2000-500649 A)    Patent Document 3: JP 2005-112827 A    Patent Document 4: WO 03/080655 (JP 2005-538693 A)    Non Patent Document 1: Nilsson B et al., Protein engineering, 1987, vol. 1(2), 107-113